Optimization of parameters affecting on CHO cell culture producing recombinant erythropoietin

Abstract

Several factors may affect erythropoietin (EPO) sugar structures including designing cell culture procedure, pH, concentration of additives, dissolved oxygen, and other physicochemical parameters. In this study, we investigated the influence of changes in effective parameters and compounds on the growth rate of Chinese hamster ovary cell (CHO) cells producing recombinant EPO. Cell culture was performed at different temperature, buffering conditions, and varied concentrations of additives such as pyruvic acid, insulin, GlutaMAX, and sodium butyrate. Results indicated that the optimal temperature and pH were 37?°C and 7.2, respectively. Also, optimal concentrations for pyruvic acid, butyrate, glutamate, and insulin were obtained to be 20?mM, 1?mM, 2?mM, and 40?μg/mL, respectively. Then, cell culture was performed in microcarrier-coated spinner flasks under the optimized condition. The results showed recombinant human EPO (rhEPO) production with adequate purity. Optimization of physicochemical conditions and culture media are important factors to improve the quantity and quality of protein products. This study showed that cell growth and recombinant EPO protein production significantly increased under the optimized conditions. The results of this research can also be used in scale-up to increase the efficiency of EPO production.

Abbreviations: EPO: erythropoietin; CHO cell: Chinese hamster ovary cell; rhEPO: recombinant human EPO; DMEM: modified eagle’s medium; FBS: fetal bovine serum; SDS-PAGE: sodium dodecyl sulfate–polyacrylamide gel electrophoresis; IGF-1: insulin-like growth factor 1     

A Dermal Gel Made of Rutilus Kutum Skin Collagen-Chitosan for Deep Burn Healing

International Journal of Peptide Research and Therapeutics - Natural compounds extracted from marine organisms consisting of biological active materials like collagen provide a major source of...     

Comparing the frequency of CD33+ pSTAT3+ myeloid-derived suppressor cells and IL-17+ lymphocytes in patients with prostate cancer and benign prostatic hyperplasia

Prostate cancer (PCa) is one of the most epidemic types of cancer in men. The tumor microenvironment (TME) of PCa is involved in the emergence of immunosuppressive factors such as myeloid-derived suppressor cells (MDSC), which regulate the immune system by several mechanisms, including interleukin (IL)-10 production. On the other hand, IL-17⁺ helper T cells (Th17) induce MDSCs and chronic inflammation in TME by producing IL-17. This study demonstrated that the frequency of CD33⁺ pSTAT3⁺ MDSC and IL-17⁺ lymphocyte as well as IL-10 messenger RNA (mRNA) expression were significantly higher in the PCa patients than in the benign prostatic hyperplasia (BPH) group. Moreover, there was no significant relationship between the frequency of CD33⁺ pSTAT3⁺ MDSC, and IL-17⁺ lymphocyte with Gleason scores in the PCa group. We suggested that the higher frequency of CD33⁺ pSTAT3⁺ MDSC and IL-17⁺ lymphocyte and the more frequent expression of IL-10 mRNA in PCa patients may play roles in tumor progression from BPH to PCa.     

Adipose tissue-derived stem cells upon decellularized ovine small intestine submucosa for tissue regeneration: An optimization and comparison method

The extracellular matrix of different mammalian tissues is commonly used as scaffolds in the field of tissue engineering. One of these tissues, which has frequently been studied due to its structural and biological features, is the small intestine submucosal membrane. These research are mainly done on the porcine small intestine. However, a report has recently been published about a scaffold produced from the submucosal layer of the ovine small intestine. In the present study, ovine small intestine submucosal (OSIS) was decellularized in a modified manner and its histological, morphological, and biomechanical properties were studied. Decellularization was performed in two phases: physical and chemical. In this method, a chloroform-methanol mixture, enzymatic digestion, and a constant dose of sodium dodecyl sulfate (SDS) was used in the least agitation time and its histological property and biocompatibility were evaluated in the presence of adipose tissue-derived stem cells (ADSCs); furthermore, ADSCs were isolated with a simple method (modified physical washing non-enzymatic isolation). The results were showed that the use of OSIS could be effective and operative. Mechanical properties, histological structure and shape, and glycosaminoglycan content were preserved. In the SDS-treated group, more than 90% of the native cells of tissue were deleted, and also in this group, no toxicity was observed and cell proliferation was supported, compared to the untreated group. Therefore, our results indicate that ADSCs seeded on OSIS scaffold could be used as a new approach in regenerative medicine as hybrid or hydrogel application.     

Centaurea shahuensis and C. ravansarensis spp. nov. (Asteraceae,Cardueae) from west Iran

Two new species of Centaurea L. sect. Cynaroides Boiss. ex Walp. (Asteraceae), C. shahuensis Ranjbar and Negaresh and C. ravansarensis Ranjbar and Negaresh are described and illustrated from Kermanshah Province, west Iran. They are closely related to C. regia Boiss. subsp. regia. However, C. shahuensis differs from it by median stem leaves broadly oblanceolate or subpandurate, phyllaries densely lanate‐tomentose, appendages small, concealing a minor part of phyllaries, and also median appendage margin entire sometimes with 1–2 cilia, 1.2–3.0 mm long on each side. Centaurea ravansarensis is distinguished by upper stem leaves loosely arachnoid, phyllaries loosely floccose‐tomentose, inner appendages deep brown to blackish, and spine 4.5–6.0 mm long. Habitat, conservation status and the geographical distribution of the new species are given.     

Evaluation of the effects of weak and moderate static magnetic fields on the characteristics of human low density lipoprotein in vitro

It has been suggested that exposure to electromagnetic fields may be a risk factor for cardiovascular disease in humans. Low density lipoprotein (LDL) modifications such as peroxidation and aggregation have been implicated in the pathogenesis of atherosclerosis. The present study investigated the effects of weak (0.125–0.5 mT) and moderate (1–4 mT) static magnetic fields (SMFs) on LDL oxidation, aggregation and zeta potential in vitro. Our results demonstrated that magnetic flux densities of 0.25 and 0.5 mT decreased, and magnetic flux densities of 3 and 4 mT increased the zeta potential and LDL oxidation in comparison with the control samples. All doses of SMFs increased the LDL aggregation in a time‐ and dose‐dependent manner. It is concluded that SMFs can alter the susceptibility of LDL to oxidation and this alteration is dependent on the applied magnetic flux density. The SMF, in addition to its role in the production and stabilization of free radicals and promotion of lipid peroxidation, may influence the metabolism of lipoproteins and their interaction with other molecules such as apolipoproteins, enzymes and receptors through the alteration of the LDL zeta potential and its particles tendency to aggregation. Bioelectromagnetics 34:397–404, 2013. © 2012 Wiley Periodicals, Inc.     

Improved production of 3‐hydroxypropionaldehyde by complex formation with bisulfite during biotransformation of glycerol

3‐Hydroxypropionaldehyde (3HPA) is an important specialty chemical which can be produced from glycerol using resting cells of Lactobacillus reuteri. This biocatalytic route, however, suffers from substrate‐ and product‐mediated loss of enzyme activity within 2 h of biotransformation. In order to overcome the inhibitory effects of 3HPA, complex formation with sodium bisulfite was investigated, optimized and applied for in situ capture of the aldehyde during biotransformation of glycerol in a fed‐batch process. As a result, the activity of the cells was maintained for at least 18 h. The 3HPA produced per gram cell dry weight was increased 5.7 times compared to the batch production process, and 2.2 times compared to fed‐batch process without in situ complex formation. This approach may have potential for production and in situ removal of 3HPA after further process development. Biotechnol. Bioeng. 2013; 110: 1243–1248. © 2012 Wiley Periodicals, Inc.     

Synthesis of novel azo compounds containing 5(4H)-oxazolone ring as potent tyrosinase inhibitors

Six new azo dyes containing of 5(4H)-oxazolone ring were prepared by diazotization of 4-aminohippuric acid and coupling with N,N-dimethylaniline, 1-naphthol and 2-naphthol and condensation with 4-fluoro benzaldehyde or 4-trifluoromethoxy benzaldehyde. The new compounds were fully characterized by spectroscopic techniques. All synthesized compounds exhibited high tyrosinase inhibitory behavior. The results of mushroom tyrosinase inhibition assays indicate that the 4-trifluoromethoxy derivatives have high degrees of inhibition and N,N-dimethylaniline derivatives are better for tyrosinase inhibition than 1-naphthol and 2-naphthol derivatives. All synthesized azo compounds (4a–4f) showed the most potent mushroom tyrosinase inhibition, comparable to that of Kojic acid and l-mimosine, as reference standard inhibitors.